Simultaneous Replacement of Endothelial Thrombomodulin and Plasma Protein C: A Novel Therapeutic Strategy for Sepsis-Induced Disseminated Intravascular Coagulation

Sepsis induces a procoagulant state, which in its most extreme form can result in disseminated intravascular coagulation (DIC), a syndrome of widespread microvascular thrombosis, ischemia, and organ dysfunction. Suppression of the protein C (PC) pathway has long been thought to be an important part of the pathophysiology, leading to development of a number of candidate therapeutics, including soluble human thrombomodulin (shTM, or ART123), currently being evaluated in a phase III clinical trial. While conferring less risk of bleeding toxicity than previous pharmacologic interventions into the protein C pathway, shTM fails to reproduce the spatial localization of its endogenous counterpart, preventing optimal interaction with PC bound to its endothelial receptor. To test the importance of localization to the endothelial membrane, we compared the antithrombotic activity of shTM to that of hTM/R6.5, a fusion protein therapeutic that anchors recombinant human TM to endothelial ICAM-1. In modified thrombin generation assays, in which platelet-poor plasma was exposed to cytokine-activated human endothelial cells, cell bound hTM/R6.5 was more potent than shTM mixed into the plasma. A similar result was found in a human whole blood microfluidic assay of sepsis-induced microvascular thrombosis, in which endothelial bound hTM/R6.5 more effectively inhibited fibrin deposition than its soluble counterpart. To investigate its mechanism of action, the fusion protein was tested in the setting of moderate PC deficiency, a common occurrence in patients with DIC. While PC depletion alone had no effect on fibrin deposition in this model, it significantly reduced the efficacy of hTM/R6.5, which was restored by supplementation using plasma-derived PC concentrate. These results indicate that endothelial bound TM acts not only as a direct inhibitor of thrombin, but derives antithrombotic activity from the activation of PC, such that it may be insufficient to reverse the coagulopathy of patients with significant deficiency in plasma PC. Likewise, the data match the clinical experience with PC supplementation, which appears to be of nominal benefit in sepsis or DIC, despite repeated demonstration of efficacy in patients with severe congenital PC deficiency, who have normal levels of endothelial TM and EPCR. The synergistic effect observed with simultaneous replacement of plasma PC and endothelial TM, however, suggest that this combination may represent a novel, translational therapeutic strategy.

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(a) Thrombin generation assay using fluorescent thrombin substrate. Thrombin was generated following addition of platelet poor plasma to static monolayers of tissue factor expressing, TNF-α activated HUVEC. Treatment of cells with hTM/R6.5, followed by washing to remove non-specifically bound fusion protein, was more effective than addition of shTM to the plasma. Inset shows AUC (area under curve) analysis: * = p < 0.05 vs. TNF only, # = p < 0.05 for hTM/R6.5 vs. shTM at each concentration. (b) Fibrin generation (measured using AF647-conjugated fibrin antibody) in whole blood microfluidic assay. 3D-confluent endothelial monolayers were grown within flow chambers and activated with TNF-α prior to the infusion of whole blood at 5 dynes/cm². hTM/R6.5 shows more prolonged inhibition of coagulation than shTM. Inset shows fluorescence microscopy images at 30 min after infusion of whole blood and AUC analysis: * = p < 0.05 vs. TNF only. (c) Fibrin deposition in control vs. PC deficient blood. PC supplementation with plasma-derived concentrate restores the anti-thrombotic activity of hTM/R6.5. Inset shows fluorescence microscopy images for each condition at 7.5 min after infusion of whole blood. AUC analysis is also shown: ** = p < 0.05 vs. all other conditions.

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(a) Thrombin generation assay using fluorescent thrombin substrate. Thrombin was generated following addition of platelet poor plasma to static monolayers of tissue factor expressing, TNF-α activated HUVEC. Treatment of cells with hTM/R6.5, followed by washing to remove non-specifically bound fusion protein, was more effective than addition of shTM to the plasma. Inset shows AUC (area under curve) analysis: * = p < 0.05 vs. TNF only, # = p < 0.05 for hTM/R6.5 vs. shTM at each concentration. (b) Fibrin generation (measured using AF647-conjugated fibrin antibody) in whole blood microfluidic assay. 3D-confluent endothelial monolayers were grown within flow chambers and activated with TNF-α prior to the infusion of whole blood at 5 dynes/cm². hTM/R6.5 shows more prolonged inhibition of coagulation than shTM. Inset shows fluorescence microscopy images at 30 min after infusion of whole blood and AUC analysis: * = p < 0.05 vs. TNF only. (c) Fibrin deposition in control vs. PC deficient blood. PC supplementation with plasma-derived concentrate restores the anti-thrombotic activity of hTM/R6.5. Inset shows fluorescence microscopy images for each condition at 7.5 min after infusion of whole blood. AUC analysis is also shown: ** = p < 0.05 vs. all other conditions.

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